Development of an automated fluorescence microscopy system for photomanipulation of genetically encoded photoactivatable proteins (optogenetics) in live cells

Abstract

Photomanipulation of genetically encoded light-sensitive protein activity, also known as optogenetics, is one of the most innovative recent microscopy techniques in the fields of cell biology and neurobiology. Although photomanipulation is usually performed by diverting the photobleaching mode of a confocal laser microscope, photobleaching by the laser scanning unit is not always suitable for photoactivation. We have developed a simple automated wide-field fluorescence microscopy system for the photomanipulation of genetically encoded photoactivatable proteins in live cells. An electrically automated fluorescence microscope can be controlled through MetaMorph imaging software, making it possible to acquire time-lapse, multiwavelength images of live cells. Using the journal (macro recording) function of MetaMorph, we wrote a macro program to change the excitation filter for photoactivation and illumination area during the intervals of image acquisition. When this program was run on the wide-field fluorescence microscope, cells expressing genetically encoded photoactivatable Rac1, which is activated under blue light, showed morphological changes such as lamellipodial extension and cell surface ruffling in the illuminated region. Using software-based development, we successfully constructed a fully automated photoactivation microscopy system for a mercury lamp-based fluorescence microscope.

Rab10-Positive Tubular Structures Represent a Novel Endocytic Pathway That Diverges From Canonical Macropinocytosis in RAW264 Macrophages

“…The optogenetic photo-manipulation of PA-Rac1 activity was performed as previously described (9,20). Briefly, cells were transfected with pTriEx/mCherry-or pECFP-PA-Rac1 ( 21) and observed under the LSM700 confocal microscope controlled by ZEN (Zeiss) By illuminating a blue-light laser (445 or 488 nm wavelength) to the cells expressing PA-Rac1 under the indicated conditions, PA-Rac1 was activated through the conformational change of the light oxygen voltage 2 domain (LOV2) of PA-Rac1 in either a local area or the whole-cell region.…”

“…To observe the dynamics of a red fluorescent protein-tagged protein during PA-Rac1 photo-manipulation, pECFP-PA-Rac1 was employed. In some optogenetic photo-manipulation experiments were performed using the Leica DMI6000B automated epifluorescence microscopy system, controlled by MetaMorph software (Molecular Devices) (20). The acquired fluorescence images were processed using the Safir denoising software (INRIA) and the nearest neighbor deconvolution algorism of the MetaMorph software.…”

Small GTPases and phosphoinositides in the regulatory mechanisms of macropinosome formation and maturation

“…Markers of macropinosome maturation, such as PI(3)P and Rab21, were recruited to the formed macropinosomes after the irradiation was turned off (Figure 2C). However, when we continued the irradiation, macropinosome formation was not observed; cup or pocket-like structures instead accumulated at the photoactivation region (Fujii et al, 2013; Araki et al, 2014). Thus, Rac1 activation alone is insufficient for closing macropinocytic cups into macropinosomes, although it efficiently forms circular ruffles.…”

Invention of a novel photodynamic therapy for tumors using a photosensitizing PI3K inhibitor

“…Photoactivation and live‐cell imaging were performed using a Leica DMI 6000B inverted microscope operated with a MetaMorph imaging system (Molecular Devices, Sunnyvale, CA). We automated the microscope for photoactivation using the macroprogramming capability of the MetaMorph software . An external light source (Leica EL6000) with a high‐pressure short‐arc mercury lamp (Osram HXP R 120 W/45C VIS) was used for fluorescence excitation.…”

“…We automated the microscope for photoactivation using the macroprogramming capability of the MetaMorph software. 25 An external light source (Leica EL6000) with a high-pressure short-arc mercury lamp (Osram HXP R 120 W/45C VIS) was used for fluorescence excitation. We mainly used a 430-nm cyan fluorescent protein (CFP) excitation filter (ET-ECFP; Chroma Technology, Bellows Falls, VT) to photosensitize XL147.…”