Scientific Report: Fluorescence Microscopy and Cell Death Analysis

This scientific report details an experiment using fluorescence microscopy to image cellular components and monitor cell death. HeLa cells were stained with Hoechst 33342, GFP, and Mitotracker Orange to visualize the nucleus, membranes, and mitochondria, respectively. U2OS cells were subjected to time-lapse fluorescence microscopy to observe apoptosis under different treatments (Camptothecin and H2O2). The study aimed to accurately image cellular components and confidently monitor cell death. While the first objective was achieved successfully, the second was partially met due to limitations with the Hoechst fluorophore. The report concludes that H2O2 induces more rapid apoptosis than Camptothecin, and suggests future studies should explore alternative fluorophores, lower concentrations, and less toxic solvents to improve the accuracy and reliability of cell death monitoring.

Advanced Microscopy and Imaging: Minor Scientific Report
Multicolor Fluorescence Analysis of Cellular Components and Surveying Cell Death by Time-Lapse Fluorescence Microscopy
Student:…………………………………………………….
Aim : 1. To image the three cellular components by Fluorescence Microscopy using fluorescent probes.
2. To monitor cell death by time-lapse fluorescence microscopy using fluorescent probes.
Results:
Figure 1: Spectra analysis of Hoechst 33342, GFP and Mitotracker Orange in relation to three different
filter sets.
The excitation spectra of the fluorophores are represented by the dotted lines and emission spectra are
represented by the solid lines. Fluorescent filter cubes are represented by the colored boxes. (Panel A)
Blue emission filter set in relation to Hoechst 33342, GFP and Mitotracker Orange, the grey box is the
excitation filter (360-380nm), the blue box is the emission filter (420-460nm). (panel B) green emission
filter set in relation to Hoechst 33342, GFP and Mitotracker orange, the cyan box is the excitation filter
(470-495nm), the green box is emission filter (510-550nm). (Panel C) Red emission filter set in relation
to Hoechst 33342, GFP and Mitotracker Orange, the green box is the excitation filter (530-550nm), the
yellow box is the emission filter (570nmLP).
Panel A: Control
Panel B: Hoechst
33342
Panel C: GFP
Panel D:
Mitotracker Orange
Panel E: All
Figure 2: Multicolour fluorescence analysis of HeLa cells at separate channels with overlayed images
Each figure was individually detected in fluorescence (blue/green/red) channels. Blue color represents
nucleus stained by Hoechst 33342 (panel B and E); green color represents membranes stained by GFP
(panel c and e); red color represents mitochondria stained by Mitotracker orange in DMSO (Panel D
and E) and overlay figures were generated by overlaying figures from all four channels into a single
composite image using FIJI. Our microscope was fitted with an XFO6 filter cube (Ex-340-390, Em-
420-460), an NIBA filter cube (Ex-470-490, Em-510-550) and lastly a NIB filter cube (Ex-470-490,
Em-510 LP) All figures were adjusted by FIJI (Fiji is just image J) software with the same standard
for each channel.
Panel A:
Auto-fluorescence
Panel B:
No treatment
Panel C: Camp
Panel D: H2O2
60 min 120 min 180 min 240 min 300 min 360 min
Figure 3: Live cell time lapse fluorescence analysis of U2OS cells
(Homo sapiens bone osteosarcoma) during apoptosis under different treatments.
Auto-fluorescence was represented as green color in U2OS cells with no-treatment
and no fluorescence dyes (panel A and Fig. 3a). U2OS cells were stained with
fluorophores Tetramethylrhodamine (TMRM, 100ng/ml in DMSO, red), Cellevent
(80um in DMSO, green) and Hoechst 33342 (5ug/ml in water, blue) treated with
no-treatment, Camptothecin (2ug/ml in water) and H2O2(2mM in water),
respectively (panel B, C and D). The time (in minutes) after exposure to treatments
is overplayed at the top of each figure.
All images were captured with Nikon Ti inverted microscope with 40X Ph
PlanFLour objective (0.6NA) and photometrics HQ2 CCD cooled camera. The
microscope was equipped with blue emission filters (ex360-380, em420-460 LP
and Ex 530-550 Em 570LP) and a green emission filter (ex470-495 em510-550).
Contrast/Brightness adjustment of all figures made by FIJI (formerly image J)
software.
References:
Jones, K., Kim, D.W., Park, J.S. and Khang, C.H., 2016. Live-cell fluorescence imaging to investigate the dynamics of plant cell death during infection by the rice blast fungus Magnaporthe oryzae. BMC plant biology, 16(1), p.69.
Mochizuki, S., Minami, E. and Nishizawa, Y., 2015. Live‐cell imaging of rice cytological changes reveals the importance of host vacuole maintenance for biotrophic invasion by blast fungus, Magnaporthe
oryzae. Microbiologyopen, 4(6), pp.952-966.
Swedlow, J.R. and Platani, M., 2002. Live cell imaging using wide-field microscopy and deconvolution. Cell structure and function, 27(5), pp.335-341.
Zamir, E., Geiger, B. and Kam, Z., 2008. Quantitative multicolor compositional imaging resolves molecular domains in cell-matrix adhesions. PLoS One, 3(4), p.e1901.
Figure 4: Time-lapse fluorescence intensity and cell count analysis of U2OS cells (Homo sapiens bone osteosarcoma) during apoptosis under treatments. Cell were stained with Hoechst (blue lines) and fluorescence
intensity of mitochondria (TMRM) (orange lines) imaged over a 360-minute time course. Treatment: None (Panel A), Treatment: Camptothecin (2ug/ml in water, Panel B) and treatment H2O2 (2mM in water, Panel C).
Average fluorescence intensity and fluorescent cell counts were generated by FIJI (Formerly image J) software.
Conclusion: The objective of using fluorescent probes is to stain the three cellular components and then image them accurately and with confidence. This objective was successfully achieved in this study. The second
objective of monitoring cell death accurately and with confidence by time lapse microscopy using fluorescent probes has been achieved partially as confidence was limited due to the fluorophore Hoechst. Although the
autofluorescence of U2OS cells had the same color with cellevent (Fig. 3 panel A), the autofluorescence intensity was too low to interfere with the assay. Further, cell apoptosis was observed in stained cells with/without
treatment. H2O2 treated cells exhibited a rapid apoptosis in comparison to Camptothecin treated cells (Fig. 3 and Fig. 4), and both treated/stained cells exhibited rapid apoptosis than the untreated/stained cells (Fig. 3).
Apoptosis of cells in untreated/stained group may be due to the toxicity of the fluorophore molecules, or the solvent (DMSO). In addition, cell apoptosis may also be induced due the high energy dissipated by the short
wavelength used to excite the Hoechst 33342. Therefore, in subsequent studies, use of an alternate fluorophore may be a tested. The following may be suggested for future studies: Selecting fluorophores that require
longer wavelengths for excitation. The fluorophore concentration may be reduced, maintaining saturation, for cell staining. Selecting an alternate solvent with lower toxicity to the cells and thus exhibit least interference
in study.
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Panel A: No Treatment
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Panel B: Camptothecin Treated