- Email: [email protected]
Real time confocal laser scanning microscopy: Potential applications in space medicine and cell biology
Acta Astronautica Vol. 42. Nos. l-8, pp. 37-50. 1998 01998 Elsevier Science Ltd. All rights reserved Printed in Great Britain OO94-5765/98 1619.OO+ 0.00
Pergamon
PII:Soo94-5765(98)00104-O
REAL TIME CONFOCAL LASER SCANNING MICROSCOPY: APPLICATIONS
POTENTIAL
IN SPACE MEDICINE AND CELL BIOLOGY
Ana Rollan, Thelma Ward and Anthony P. McHale*
Biotechnology Research Group, School of Applied Biological and Chemical Sciences, University of Ulster, Cromore Rd., Coleraine, Co. Londondeny, BT52 ISA, Northern Ireland. ABSTRACT Photodynamic therapy (PDT), in which tissues may be rendered fatally light-sensitive represents a relatively novel treatment for cancer and other disorders such as cardiovascular disease. It offers significant application to disease control in an isolated environment such as space flight. In studying PDT in the laboratory, low energy lasers such as HeNe lasers are used to activate the photosensitized cellular target. A major problem associated with these studies is that events occurring during actual exposure of the target cells to the system cannot be examined in real time. In this study HeLa cells were photosensitized and photodynamic activation was accomplished using the scanning microbeam from a confocal laser scanning microscope.
This form of activation allowed
for simultaneous photoactivation and observation and facilitated the recording of events at a microscopic level during photoactivation.
Effects of photodynamic activation on
the target cells were monitored using the fluorophores rho&mine 123 and ethidium homodiier- 1. Potential applications of these forms of analyses to space medicine and cell biology are discussed. 01998 Elsevier Science Ltd. All rights reserved Phone: +01265 324616
FAX: +01265 324906
* Author for correspondence. 31
Email: [email protected]
38
12th Man in Space Symposium
1.
INTRODUCTION In more recent times technological advances in medicine have been directed
towards the development of minimally invasive or non-invasive treatment modalities for a variety of disease states.
Some of those modalities offer many attractions to those
involved in planning long-duration space missions both from a medical and biological point of view.
In a medical context, remote medical procedures,
which could be
diagnostic or therapeutic in nature, offer many advantages in on-board health-care.
In
studies relating to cell biology, ground-based control of on-board equipment exploiting the most advanced analytical technologies available, offers significant advantages to both on-board and ground-based scientific personnel. In the context of advanced relatively non-invasive medical procedures, photodynamic therapy (PDT) has emerged as a relatively novel and effective treatment modality for cancer [l] and other disorders including cardiovascular disease [2]. The most commonIy used clinical procedure involves the systemic administration of a suitable photosensitizer preparation such as Photofiin@ II. This material is then taken up by the tumour cells and they become photosensitive.
The relevant site is then irradiated with
low energy laser radiation and the consequential formation of toxic oxygen at that site results in cell death and tumour eradication [3].
Although uptake of the photosensitizer
by tumour cells render those cells susceptible to PDT, normal tissues may also be photosensitized and, as a consequence, also become susceptible to PDT [4]. The latter phenomenon has led to investigations at a cell biology level in order to expand on the existing anti-cancer application of the technology [5,6,7].
In laboratory studies relating
I ?th Man in Space Symposium
39
to PDT. cell cultures are usually used as the target and low power lasers such as the HeNe laser are exploited as the photo-stimulant [5].
In this context, the effects that
occur after photoactivation are observed and the events that occur at the time of activation have remained elusive. However. in recent years our group has been studying the effects of photosensitizers on human erythrocytes with a view towards the development of drug targeting systems based on PDT [8,9]. During those studies it was shown that the laser microbeam
emitted by a confocal laser scanning microscope could be used to
simultaneously initiate the photodynamic activation event and to visualize the events occurring in real time during activation [IO].
It was found that the photosensitized
e@uocytes disrupted at certain times following exposure to the scanning beam and this appeared to be consistent with membrane disruption.
It was also found that the time
taken for this disruptive event to occur was related to the power output of the laser scanning beam and to the photosensitizer concentration[ lo]. In the study presented here we describe the effects of the scanning microbeam on photosensitized HeLa cells and report on the most immediate events that become visible in real time using the confocal laser scanning system together with the fluorescent probes rhodamine 123 and ethidium homodimer- 1. In reporting these results we illustrate the potential of this technique in the context of space medicine and cell biology and suggest a number of possible applications in those areas.
12th Man in Space Symposium
2.
MATERIALS AND METHODS
2.1
Cell line and growth conditions. The HeLa cell line used in this study was obtained from the European Collection
of Animal Cell Cultures (ECACC no. 85060701) and was maintained on minimal essential medium (Gibco) supplemented with Earl’s balanced salt solution (Gibco). 1% (v/v) non-essential amino acids (Gibco) and 10% (v/v) foetal bovine serum (Gibco). Cells were grown in a 5% CO, humidified atmosphere at 37°C. Harvesting of cells was carried out by centrifugation following treatment with 0.05% (w/v) trypsin and 0.02% (w/v) EDTA in phosphate buffered saline (PBS) for 5 min. at 30°C. Prior to examination using the confocal laser scanning system cells were inoculated on to microscope cover slips or plated in the wells of a 6-well tissue culture plate.
2.2
Photosensitization of cell populations. Hematoporphyrin derivative (HPD) was prepared as described previously [ 111.
Cells were photosensitized by incubation for 1 hour in medium containing HPD at a concentration of 5OOug/ml. Cells were washed twice by removal of the HPD solution and replacement with fresh medium.
2.3
Real time confocal laser scanning microscopy. Photosensitized cells were examined using a Nikon Optiphot microscope equipped
with a X50 oil immersion objective lens and a X40 water immersion objective lens. The microscope was linked to an Odyssey confocal laser scanning system (ODYSSEY, Noran
41
12th Man in Space Symposium
Instruments Ltd.. UK) with real time imaging facilities in order to observe photodynamic activation. Activation was accomplished using the visualizing laser microbeam emitted by a 300mW argon ion laser with multi-line emission at 458.488 and 529nm.
However
in the work presented here the 488~1 emission line was used. The system was equipped to
facilitate variability in power of emission striking the sample.
The system was also
equipped to visualize the sample in either reflected light mode or in fluorescent mode. The microscope and scanning system were controlled by the ODYSSEY software package loaded into a 486 IBM compatible PC, driven by Microsoft Windows MS-DOS system. A television monitor connected to the system facilitated direct viewing of events and those events could be video-taped using a conventional VCR system.
2.3
Fluorescent staining of cells. Photosensitized cells were incubated in tissue culture medium containing 2Opg/ml
rhodamine 123 (Molecular Probes, U.S.A.) or ethidium homodimer- 1 (Molecular Probes, U.S.A.).
Photoactivation was carried out using the visualizing microbeam from the
confocal system and the accumulation of the relevant fhrorophore was examined using the fluorescence mode of the confbcal system. The wavelength of the emitted beam was set at 488nm. Control samples consisted of non-photosensitized cells in the presence of the relevant fluorophore.
42
12th Man in Space Symposium
3.
RESULTS AND DISCUSSION
3.1
Uptake of rhodamine 123 during photoactivation of
photosensitized HeLa
cells. In our previous work it was found that photosensitized erythrocytes exhibited a disruptive event when viewed with the scanning beam of a real time confocal laser scanning microscope system and it has been suggested that this event occurred because photosensitizer in the membrane resulted in the breakdown of that membrane [lo].
This
event was very obvious and rapid. In studying HeLa cells using the confocal scanning system no obvious or immediate events occurred within a similar time span.
It was
therefore decided to examine the possibility of activating the photosensitizer in the presence of fluorogenic probes in order to examine the events occurring in real time.
In
the past it has been possible to use fluorogenic probes in conjunction with confocal laser scanning microscopy, but none of those studies included examination of the immediate events occurring in real time and involved the use of fixatives [ 121.
In the study
presented here it was decided to examine the uptake of rhodamine 123 by HeLa cells during photoactivation in real time. Rhodamine 123 is normally used as a mitochondrial probe and its uptake by the mitochondria is dependant on metabolic ftmction. Photosensitized HeLa cells were placed in the scanning beam in the presence of the fluorophore and the images obtained were recorded in real time. The results obtained using the reflected light channel are shown in Fig. 1 (Plates A-B) with Plate A showing a single cell at 5 min. and Plate B showing the same cell following 15min. exposure to the scanning beam.
The results do not show any major difference apart from a slight
43
12th Man in Space Symposium
1A
1D
Fig.1 Uptake of rhodamine 123 by photosensitized HeLa cells during activation by the microbeam emitted by the confocal laser scanning microscope.
All information was
recorded in real time and a single cell was viewed using reflected light operating mode (Plates A and B) or fluorescent light operating mode (Plates C and D) following 5 min. and 15 min. exposure to the emitted microbeam. scanning beam at time zero exhibited no fluorescence.
Control samples exposed to the
12th Man in Space Symposium
44
2A
28
2D
Fig.2 Uptake of ethidium homodimer- 1 by photosensitized HeLa cells during activation by the confocal laser scanning microscope microbeam.
All information was recorded in
real time and viewed using either the reflected light operating mode (Plates A & B) at 5 and 15 min. respectively or using the fluorescent light operating mode (Plates C and D) at 5 and 15 min. respectively. zero exhibited no fluorescence.
Control samples exposed to the scanning beam at time
45
12th Man in Space Symposium
rounding up of the cell with the outer cell edges moving towards the centre. The results obtained using the fluorescent channel in order to examine the uptake of the fluorophore demonstrated an initial lack of the fluorophore in the cell at 5 min. with a massive degree of uptake following 15 min. exposure to the scanning beam (Fig. 1, Plates C and D, respectively). Although this fluorophore is relatively specific for mitochondrial activity this was not apparent from the images obtained.
It has however been suggested that
photodynamic activation results in decreased mitochondrial function [3 J and this may explain the absence of any obvious fluorophore in the cells examined.
However, the
results do indicate severely compromised cell membrane function with consequential uptake of the fluorophore into the cytoplasm.
These results are the first to describe the
use of this technique in studying the events that occur during photoactivation and in real time.
The results also demonstrate the power of this technique in studying cellular
function in real time.
3.2
Uptake of ethidium homodimer- 1 by photoactivated photosensitized HeLa cells. In order to confirm the effects on membrane fL.nction observed above during
photoactivation, it was decided to cany out a similar study replacing the rhodamine 123 with ethidium homodimer- 1. Uptake by the latter is recognized to be a measure of the membrane integrity with absorption of the fluorophore occurring when that integrity is compromised [ 131. In the normal course of events the fluorophore is taken up by nuclear material.
Photosensitized erythrocytes were placed in the laser scanning beam of the
confbcal system and events occurring in real time were examined using the reflected light
12th Man in Space Symposium
channel as shown in Fig. 2 (Plates A & B).
Plate A contains an image of the cell 5 min.
and Plate B contains an image of the same cell exposed to the scanning beam for 1Smin. The images show little difference occurring over this time span. However. when the cells were examined using the fluorescent channel of the confocal system in order to detect uptake of the fluorophore, a definite increase in fluorescence occurred within the cells over this time period (Fig.2., C & D). Image C was the cell examined at 5 min. and image D was obtained following 15 min. exposure to the beam.
It should be noted
however, that the uptake of the fluorophore did not appear similar to that observed using the rhodamine 123. Possible reasons for this are currently under consideration, although non-real time imaging of photodynamic activation events do suggest an uneven uptake of the ethidium homodimer- 1. In any event the results do suggest that the integrity of the cell membrane is compromised during photoactivation and further demonstrates the value of this technique in studying events at a cellular level and in real time.
3.3
Possible use of the above technology in space medicine and cell biology.
As mentioned above the development of photodynamic therapy (PDT) has offered a variety of novel treatment modalities for a number of existing medical conditions. also been mentioned
It has
that cell death, which occurs following exposure of the
photosensitized cells to the scanning beam, may be brought about in normal tissues and this is based, simply on the presence of the photosensitizer in those tissues.
This
suggests that the technique offers unique possibilities particularly in the area of microsurgery and the removal of small quantities of unwanted tissues in a relatively non-
47
12th Man in SpaceSymposium
invasive manner. In the context of space medicine, cell or small areas of tissue could be photosensitized and removed using a microbeam.
The microbeam could be delivered
using the confocal system, possibly including the use of fibre optics for deeper sites, and the events occurring in the treated are could be monitored in real time using the confocal system. We believe that this offers vast applications to space medicine especially if the system was operated on a remote basis under the control of ground-based personnel.
The
treated area could be diagnosed as abnormal using the confocal system, and the tissue could then be photosensitized.
Treatment could then be controlled and effected by a
remote operator reducing the dependence of flight personnel on on-board medical expertise. In the context of studies relating to cell biology in space, the confocal system offers a vast array of opportunities and these include the possibility of studying individual cells in real time, at a microscopic level and using higher resolving power than conventional light microscopy.
These opportunities are further enhanced if one
considers the possible use of fluorogenic probes together with the scanning system.
In
suggesting the use of the confocal scanning system in space cell biology, it would be our opinion that remote control of the system would be achievable with some major modifications to the existing control software packages. Networking an on-board system with external ground-based control would offer multiple access to a single on-board analytical tool and further enhance the value of that analytical tool in a space environment. Although the opportunities presented by this system are multiple, problems still
48
12th Man in Space Symposwm
do exist and these include the lack of an existing networked external control system and the mass of the confocal scanning system.
We are informed by external experts that the
external control of the system is not an insurmountable problem.
However. the mass of
the existing real-time confocal system does present some problems. although in light of the currently obvious advantages with the existing system. constructing such a system with light-weight materials does offer certain possibilities.
In addition, the new two
photon systems appearing on the market (BioRad.) do offer very close to real time capabilities together with reduced mass.
4.
REFERENCES
1.
Kessel, D., Localization and photosensitization of neoplastic disease.
Trends
fhotochem. Photobiof. 1, 181-183 (1993).
2.
Shuitmaker. J.J., Bass. P.. van Leendoed, H.L.L.M.. van de Meulen. F.W., Star,
W.M. and van Zandwijk, N. Photodynamic therapy: a promising new modality for treatme of cancer. J. Photochem. Photobiol. B: Biol., 34. 3-12 (1996).
3.
Hamblin. M.R. and Newman. E.L. Photosensitizer targeting in photodynamic
therapy.
1
Conjugates of hematophorphyrin
with albumin and transferrin.
J.
Photochem. Photobiol. B: Biol. 26. 45-56 (1996).
4.
Pope, A.J. and Bown, S.G. Photodynamic therapy. Br. J. Ural. 68, l-9 ( 199 1).
12th Manin Space Symposium
5.
McHale, A.P., McHale, L. and Blau, W.
49
The effect of hematoporphyrin
derivative and human erythrocyte ghost encapsulated hematoporphyrin derivative on a mouse myeloma cell line. Cancer Biochem. Biophys.
6.
10, 157-164 (1988).
McHale, A.P. and McHale, L. Use of a tetrazolium based calorimetric assay in
assessing photoradiation therapy in vitro. Cancer Letts. 41, 3 15-32 1 (1988).
7.
Ward. T., Rollan, A., Flynn. G.. McHale. A.P. The effects of electric fields on
photosensitized
erythrocytes:
possible enhancement of photodynamic activation.
Cancer Lefts. 106, 69-74 (1996).
8.
Flynn. G., Hackett, T.J., McHale. L. and McHale, A.P.
Encapsulation of the
thrombolytic enzyme, brinase, in photosensitized erythrocytes:
A novel thrombolytic
system based on photodynamic activation. J. Photochem. Photobiol. B:Biol. 26, 193196. (1994).
9.
Flynn G., McHale, L. and McHale, A.P.
erythrocytes:
Methotrexate-loaded, photosensitized
A photoactivatable carrier/delivery system for use in cancer therapy.
Cancer Letts. 82, 225-229 (1994).
12th Man in Space Symposium
10.
Rollan, A., Ward, T., Flynn, G., McKerr, G., McHale, L. and McHale, A-P. Use
of real-time confocal laser scanning microscopy to study immediate effects of photodynamic activation on photosensitized erythrocytes.
Cancer Letts. 101, 165-169
(1996).
11.
Dougherty,
T.J.
Photo+namic
therapy.
In:
Methods in Porphyrin
Photosensitization. 3 13-323 [Ed. Kessel, D.] Plenum Press, New York (1985).
12.
Woodbum. F.W.. Vardaxis. N.J.. Hill. J.S.. Kaye, A.H. and Phillips. D.R.
Subcellular localization of porphyrins using confocal laser scanning microscopy. Photochem. Photobiol. 54. 725-732 (1991).
13.
Small. D.L.. Monette. R., Buchan. A.M. & Morley. P. Identification of calcium
channels involved in neuronal injury in rat hippocampal slices subjected to oxygen and glucose deprivation.
14.
Brain Res. 753. 209-2 18 (1997).
Wilson. B.C.. Olivo. M. and Singh, G. Subcellular localization of Photofrin and
amilnolevulinic acid and photodynamic cross resistance in vitro in radiation induced fibrosarcoma cells sensitive or resistant to Photofrin’” -mediated photodynamic therapy. Photochem. Photobiol. 65, 166-l 76 (1997).
Pergamon
PII:Soo94-5765(98)00104-O
REAL TIME CONFOCAL LASER SCANNING MICROSCOPY: APPLICATIONS
POTENTIAL
IN SPACE MEDICINE AND CELL BIOLOGY
Ana Rollan, Thelma Ward and Anthony P. McHale*
Biotechnology Research Group, School of Applied Biological and Chemical Sciences, University of Ulster, Cromore Rd., Coleraine, Co. Londondeny, BT52 ISA, Northern Ireland. ABSTRACT Photodynamic therapy (PDT), in which tissues may be rendered fatally light-sensitive represents a relatively novel treatment for cancer and other disorders such as cardiovascular disease. It offers significant application to disease control in an isolated environment such as space flight. In studying PDT in the laboratory, low energy lasers such as HeNe lasers are used to activate the photosensitized cellular target. A major problem associated with these studies is that events occurring during actual exposure of the target cells to the system cannot be examined in real time. In this study HeLa cells were photosensitized and photodynamic activation was accomplished using the scanning microbeam from a confocal laser scanning microscope.
This form of activation allowed
for simultaneous photoactivation and observation and facilitated the recording of events at a microscopic level during photoactivation.
Effects of photodynamic activation on
the target cells were monitored using the fluorophores rho&mine 123 and ethidium homodiier- 1. Potential applications of these forms of analyses to space medicine and cell biology are discussed. 01998 Elsevier Science Ltd. All rights reserved Phone: +01265 324616
FAX: +01265 324906
* Author for correspondence. 31
Email: [email protected]
38
12th Man in Space Symposium
1.
INTRODUCTION In more recent times technological advances in medicine have been directed
towards the development of minimally invasive or non-invasive treatment modalities for a variety of disease states.
Some of those modalities offer many attractions to those
involved in planning long-duration space missions both from a medical and biological point of view.
In a medical context, remote medical procedures,
which could be
diagnostic or therapeutic in nature, offer many advantages in on-board health-care.
In
studies relating to cell biology, ground-based control of on-board equipment exploiting the most advanced analytical technologies available, offers significant advantages to both on-board and ground-based scientific personnel. In the context of advanced relatively non-invasive medical procedures, photodynamic therapy (PDT) has emerged as a relatively novel and effective treatment modality for cancer [l] and other disorders including cardiovascular disease [2]. The most commonIy used clinical procedure involves the systemic administration of a suitable photosensitizer preparation such as Photofiin@ II. This material is then taken up by the tumour cells and they become photosensitive.
The relevant site is then irradiated with
low energy laser radiation and the consequential formation of toxic oxygen at that site results in cell death and tumour eradication [3].
Although uptake of the photosensitizer
by tumour cells render those cells susceptible to PDT, normal tissues may also be photosensitized and, as a consequence, also become susceptible to PDT [4]. The latter phenomenon has led to investigations at a cell biology level in order to expand on the existing anti-cancer application of the technology [5,6,7].
In laboratory studies relating
I ?th Man in Space Symposium
39
to PDT. cell cultures are usually used as the target and low power lasers such as the HeNe laser are exploited as the photo-stimulant [5].
In this context, the effects that
occur after photoactivation are observed and the events that occur at the time of activation have remained elusive. However. in recent years our group has been studying the effects of photosensitizers on human erythrocytes with a view towards the development of drug targeting systems based on PDT [8,9]. During those studies it was shown that the laser microbeam
emitted by a confocal laser scanning microscope could be used to
simultaneously initiate the photodynamic activation event and to visualize the events occurring in real time during activation [IO].
It was found that the photosensitized
e@uocytes disrupted at certain times following exposure to the scanning beam and this appeared to be consistent with membrane disruption.
It was also found that the time
taken for this disruptive event to occur was related to the power output of the laser scanning beam and to the photosensitizer concentration[ lo]. In the study presented here we describe the effects of the scanning microbeam on photosensitized HeLa cells and report on the most immediate events that become visible in real time using the confocal laser scanning system together with the fluorescent probes rhodamine 123 and ethidium homodimer- 1. In reporting these results we illustrate the potential of this technique in the context of space medicine and cell biology and suggest a number of possible applications in those areas.
12th Man in Space Symposium
2.
MATERIALS AND METHODS
2.1
Cell line and growth conditions. The HeLa cell line used in this study was obtained from the European Collection
of Animal Cell Cultures (ECACC no. 85060701) and was maintained on minimal essential medium (Gibco) supplemented with Earl’s balanced salt solution (Gibco). 1% (v/v) non-essential amino acids (Gibco) and 10% (v/v) foetal bovine serum (Gibco). Cells were grown in a 5% CO, humidified atmosphere at 37°C. Harvesting of cells was carried out by centrifugation following treatment with 0.05% (w/v) trypsin and 0.02% (w/v) EDTA in phosphate buffered saline (PBS) for 5 min. at 30°C. Prior to examination using the confocal laser scanning system cells were inoculated on to microscope cover slips or plated in the wells of a 6-well tissue culture plate.
2.2
Photosensitization of cell populations. Hematoporphyrin derivative (HPD) was prepared as described previously [ 111.
Cells were photosensitized by incubation for 1 hour in medium containing HPD at a concentration of 5OOug/ml. Cells were washed twice by removal of the HPD solution and replacement with fresh medium.
2.3
Real time confocal laser scanning microscopy. Photosensitized cells were examined using a Nikon Optiphot microscope equipped
with a X50 oil immersion objective lens and a X40 water immersion objective lens. The microscope was linked to an Odyssey confocal laser scanning system (ODYSSEY, Noran
41
12th Man in Space Symposium
Instruments Ltd.. UK) with real time imaging facilities in order to observe photodynamic activation. Activation was accomplished using the visualizing laser microbeam emitted by a 300mW argon ion laser with multi-line emission at 458.488 and 529nm.
However
in the work presented here the 488~1 emission line was used. The system was equipped to
facilitate variability in power of emission striking the sample.
The system was also
equipped to visualize the sample in either reflected light mode or in fluorescent mode. The microscope and scanning system were controlled by the ODYSSEY software package loaded into a 486 IBM compatible PC, driven by Microsoft Windows MS-DOS system. A television monitor connected to the system facilitated direct viewing of events and those events could be video-taped using a conventional VCR system.
2.3
Fluorescent staining of cells. Photosensitized cells were incubated in tissue culture medium containing 2Opg/ml
rhodamine 123 (Molecular Probes, U.S.A.) or ethidium homodimer- 1 (Molecular Probes, U.S.A.).
Photoactivation was carried out using the visualizing microbeam from the
confocal system and the accumulation of the relevant fhrorophore was examined using the fluorescence mode of the confbcal system. The wavelength of the emitted beam was set at 488nm. Control samples consisted of non-photosensitized cells in the presence of the relevant fluorophore.
42
12th Man in Space Symposium
3.
RESULTS AND DISCUSSION
3.1
Uptake of rhodamine 123 during photoactivation of
photosensitized HeLa
cells. In our previous work it was found that photosensitized erythrocytes exhibited a disruptive event when viewed with the scanning beam of a real time confocal laser scanning microscope system and it has been suggested that this event occurred because photosensitizer in the membrane resulted in the breakdown of that membrane [lo].
This
event was very obvious and rapid. In studying HeLa cells using the confocal scanning system no obvious or immediate events occurred within a similar time span.
It was
therefore decided to examine the possibility of activating the photosensitizer in the presence of fluorogenic probes in order to examine the events occurring in real time.
In
the past it has been possible to use fluorogenic probes in conjunction with confocal laser scanning microscopy, but none of those studies included examination of the immediate events occurring in real time and involved the use of fixatives [ 121.
In the study
presented here it was decided to examine the uptake of rhodamine 123 by HeLa cells during photoactivation in real time. Rhodamine 123 is normally used as a mitochondrial probe and its uptake by the mitochondria is dependant on metabolic ftmction. Photosensitized HeLa cells were placed in the scanning beam in the presence of the fluorophore and the images obtained were recorded in real time. The results obtained using the reflected light channel are shown in Fig. 1 (Plates A-B) with Plate A showing a single cell at 5 min. and Plate B showing the same cell following 15min. exposure to the scanning beam.
The results do not show any major difference apart from a slight
43
12th Man in Space Symposium
1A
1D
Fig.1 Uptake of rhodamine 123 by photosensitized HeLa cells during activation by the microbeam emitted by the confocal laser scanning microscope.
All information was
recorded in real time and a single cell was viewed using reflected light operating mode (Plates A and B) or fluorescent light operating mode (Plates C and D) following 5 min. and 15 min. exposure to the emitted microbeam. scanning beam at time zero exhibited no fluorescence.
Control samples exposed to the
12th Man in Space Symposium
44
2A
28
2D
Fig.2 Uptake of ethidium homodimer- 1 by photosensitized HeLa cells during activation by the confocal laser scanning microscope microbeam.
All information was recorded in
real time and viewed using either the reflected light operating mode (Plates A & B) at 5 and 15 min. respectively or using the fluorescent light operating mode (Plates C and D) at 5 and 15 min. respectively. zero exhibited no fluorescence.
Control samples exposed to the scanning beam at time
45
12th Man in Space Symposium
rounding up of the cell with the outer cell edges moving towards the centre. The results obtained using the fluorescent channel in order to examine the uptake of the fluorophore demonstrated an initial lack of the fluorophore in the cell at 5 min. with a massive degree of uptake following 15 min. exposure to the scanning beam (Fig. 1, Plates C and D, respectively). Although this fluorophore is relatively specific for mitochondrial activity this was not apparent from the images obtained.
It has however been suggested that
photodynamic activation results in decreased mitochondrial function [3 J and this may explain the absence of any obvious fluorophore in the cells examined.
However, the
results do indicate severely compromised cell membrane function with consequential uptake of the fluorophore into the cytoplasm.
These results are the first to describe the
use of this technique in studying the events that occur during photoactivation and in real time.
The results also demonstrate the power of this technique in studying cellular
function in real time.
3.2
Uptake of ethidium homodimer- 1 by photoactivated photosensitized HeLa cells. In order to confirm the effects on membrane fL.nction observed above during
photoactivation, it was decided to cany out a similar study replacing the rhodamine 123 with ethidium homodimer- 1. Uptake by the latter is recognized to be a measure of the membrane integrity with absorption of the fluorophore occurring when that integrity is compromised [ 131. In the normal course of events the fluorophore is taken up by nuclear material.
Photosensitized erythrocytes were placed in the laser scanning beam of the
confbcal system and events occurring in real time were examined using the reflected light
12th Man in Space Symposium
channel as shown in Fig. 2 (Plates A & B).
Plate A contains an image of the cell 5 min.
and Plate B contains an image of the same cell exposed to the scanning beam for 1Smin. The images show little difference occurring over this time span. However. when the cells were examined using the fluorescent channel of the confocal system in order to detect uptake of the fluorophore, a definite increase in fluorescence occurred within the cells over this time period (Fig.2., C & D). Image C was the cell examined at 5 min. and image D was obtained following 15 min. exposure to the beam.
It should be noted
however, that the uptake of the fluorophore did not appear similar to that observed using the rhodamine 123. Possible reasons for this are currently under consideration, although non-real time imaging of photodynamic activation events do suggest an uneven uptake of the ethidium homodimer- 1. In any event the results do suggest that the integrity of the cell membrane is compromised during photoactivation and further demonstrates the value of this technique in studying events at a cellular level and in real time.
3.3
Possible use of the above technology in space medicine and cell biology.
As mentioned above the development of photodynamic therapy (PDT) has offered a variety of novel treatment modalities for a number of existing medical conditions. also been mentioned
It has
that cell death, which occurs following exposure of the
photosensitized cells to the scanning beam, may be brought about in normal tissues and this is based, simply on the presence of the photosensitizer in those tissues.
This
suggests that the technique offers unique possibilities particularly in the area of microsurgery and the removal of small quantities of unwanted tissues in a relatively non-
47
12th Man in SpaceSymposium
invasive manner. In the context of space medicine, cell or small areas of tissue could be photosensitized and removed using a microbeam.
The microbeam could be delivered
using the confocal system, possibly including the use of fibre optics for deeper sites, and the events occurring in the treated are could be monitored in real time using the confocal system. We believe that this offers vast applications to space medicine especially if the system was operated on a remote basis under the control of ground-based personnel.
The
treated area could be diagnosed as abnormal using the confocal system, and the tissue could then be photosensitized.
Treatment could then be controlled and effected by a
remote operator reducing the dependence of flight personnel on on-board medical expertise. In the context of studies relating to cell biology in space, the confocal system offers a vast array of opportunities and these include the possibility of studying individual cells in real time, at a microscopic level and using higher resolving power than conventional light microscopy.
These opportunities are further enhanced if one
considers the possible use of fluorogenic probes together with the scanning system.
In
suggesting the use of the confocal scanning system in space cell biology, it would be our opinion that remote control of the system would be achievable with some major modifications to the existing control software packages. Networking an on-board system with external ground-based control would offer multiple access to a single on-board analytical tool and further enhance the value of that analytical tool in a space environment. Although the opportunities presented by this system are multiple, problems still
48
12th Man in Space Symposwm
do exist and these include the lack of an existing networked external control system and the mass of the confocal scanning system.
We are informed by external experts that the
external control of the system is not an insurmountable problem.
However. the mass of
the existing real-time confocal system does present some problems. although in light of the currently obvious advantages with the existing system. constructing such a system with light-weight materials does offer certain possibilities.
In addition, the new two
photon systems appearing on the market (BioRad.) do offer very close to real time capabilities together with reduced mass.
4.
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