Light exposure and cell viability in fluorescence microscopy

Abstract

SummaryTest systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm −2 and about 300 J cm −2 for native cells (corresponding to about 250−3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi-or total internal reflection illumination) and decreased to values between 50 J cm −2 and less than 1 J cm −2 upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high-resolution wide field or laser scanning microscopy of single cells, typically 10−20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in superresolution microscopy based on single molecule detection.

A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

“…Fluorescent labels that are excited and emit in the near-infrared are especially biocompatible, because they avoid the use of light, which may cause phototoxicity or unwanted autofluorescent background 2 . Although numerous synthetic near-infrared fluorophores with exceptional photostability and brightness exist, they tend to be membrane impermeable and show unspecific binding to cellular components (Supplementary Table S1 and Fig.…”

Nanosecond ratio imaging of redox states in tumor cell spheroids using light sheet-based fluorescence microscopy

“…3). Assuming a light dose of ∼10 J∕cm 2 to be nonphototoxic for GFP mutants, 7 this implies that at least up to 200 s light-induced cell damage can probably be excluded. 7 In Fig.…”

“…Assuming a light dose of ∼10 J∕cm 2 to be nonphototoxic for GFP mutants, 7 this implies that at least up to 200 s light-induced cell damage can probably be excluded. 7 In Fig. 4, the ratio I 391 ∕I 470 of oxidized versus reduced Grx1-roGFP2 is depicted for U251MG-L106-RS and U251MG-L106-MRS glioblastoma cells before and after treatment with the apoptosis-inducing agent staurosporine applied for 5 h at a concentration of 12 μM.…”

“…This, however, often requires high light doses and risks to damage living specimens when several planes are measured successively. 7 Phototoxicity and cell damage can be avoided efficiently by light sheet-based fluorescence microscopy or single-plane illumination microscopy (SPIM), where planes under investigation are illuminated selectively in a direction perpendicular to the observation path. 8,9 Although only two commercial light sheet microscopes have been reported so far (Lightsheet Z.1, Carl Zeiss AG, Jena, Germany; Ultramicroscope, LaVision GmbH, Bielefeld, Germany), several laboratory apparatus have meanwhile been established (see, e.g., Refs.…”

Tumor cell differentiation by label-free fluorescence microscopy

“…This dose corresponds to 250 to 1000 s of solar illumination and may be easily exceeded in 3-D microscopy. 17,32 Therefore, in fluorescence or Raman microscopy low irradiance has to be combined with highly sensitive detection units, e.g., EM-CCD cameras, image intensifying systems, or very sensitive spectroscopic devices.…”