「様式３」P-65

Development of a multi-beam spectroscopic confocal
fluorescence lifetime imaging microscope［１］Organization
Project Leader：Qiyin Fang
(McMaster University)
Representative at RIE：Keiichiro Kagawa（Shizuoka University）［２］Research Progress
In this project, we studied the feasibility ofthe time-resolved CMOS image sensors
developed at Shizuoka University to compact andpotentially low-cost multiplexed confocal
fluorescence lifetime imaging (FLIM) microscopes.
One of the most time-consuming stages of drug
discovery is in the identification of drug leads.
Modernhigh-content
screening(HCS)microscopes process hundreds of thousands ofsamples, which have been treated with varied
dosages of countless drug leads. Protein-protein
interactions, such as a procaspase being cleaved
in two, or a multiple proteins forming an oligomer,
happen at a scale far below the optical diffraction
limit. This makes such interactions invisible inmorphological high-content screening. There
stands to be a great deal of insight should suchinteractions be monitored, however, since thiswould allow the determination of the exact

Fig. 1: Sample reconstructed images of multifocal
scanning setup. left: Fixed Convallaria majalis
slide right: MCF-7 breast cancer cell line taggedwith mCerulean-3 fluorophore on protein Bcl-XL.

pathway that a candidate drug is modulating.
Förster resonance energy transfer (FRET) offers
this ability. Fluorescence lifetime imaging
microscopy is suitable for measuring FRET(FLIM-FRET), where the presence of FRET canbe measured as a reduction in the fluorescence
lifetime of the donor fluorophore, making it amore accurate measure of FRET activity – and soprotein-protein interaction – within the cell.
Confocal (or multiphoton) scanning fluorescencemicroscopy is still a base requirement formeasuring protein-protein interactions by
FLIM-FRET. While laser scanning can beperformed very quickly and accurately, the timeto acquire a confocal scan is slowed down by thecollection time: the pixel dwell time must be set
such that sufficient emitted light is collected ateach position. While the laser power could beincreased, in order to reduce the required dwelltime, very high laser irradiance could lead tophotobleaching and photodamage of the specimen.
By employing multiple foci to scan across thesample, each focal spot could be provided with theoptimal laser power that would maximize thesignal without causing photodamage. The
imaging speed of a system like this would thenscale with the number of excitation foci.
Thesystemhas
beenpreviouslyimplemented with a scientific CCD imager, where
each foci is identified as an ROI on the readoutframe. Some of the results of these scans aredisplayed in Fig. 1. In these cases, a 10x10 fociarray was raster scanned with 30x30 steps on thesample, resulting in 300x300 reconstructions.
While these sample reconstructions are onlyintensity images, they show the imaging
capabilities that can be made time-resolved witha suitable detector array. Combined, the promiseof high-speed FLIM imaging for HCS drug
discovery applications appears to be very close toreality.
The streak camera can be an option to
implement time-resolving imaging. However, it isFig. 2: Experimental setup of multifocal confocal scanning microscope.

［３］Results(３－１) Research results
In order to provide a high-throughput FLIM
system, a multifocal scanning system isimplemented. Instead of a spinning disk
approach, the foci are generated by a lenslet array,and a unique scanner is implemented touniformly scan the foci array across the sample.
The scanner reverses the scan for the returning
fluorescent light, such that the fluorescent fociremain stationary, regardless of the current
scanning position on the sample. This allows forof a stationary detector array with sparsely
arranged pixels to be used for detection. The
experimental setup is shown in Fig. 2.
By the advent of multiplexing, each foci isable to independently scan its subregion of thesample, and the final resulting image will stitch
each foci’s subregion together. The system rasterscans the foci array across the sample, andcollects one frame for each scan position. If there
are MxM foci, and an NxN scan is performed, theresulting reconstructed image will be MNxMN indimension. A sparse array detector is optimal inthis case, due to the small number of foci(maximum 32x32) and the requirement for
high-speed readout in order to achieve
high-throughput screening.

LEFM driver
Read ctrl.

bulky, expensive, and difficult to scale upproduction. In this project, feasibility of Shizuoka
University’s time-resolving CMOS image senor tomulti-beam confocal FLIM has been studied. We
had meetings on May 22, 2016 and Nov. 9, 2016
for discussions.

Pixelarray(27x27)
Pitch conversion matrix
LEFM driver

Readout
circuits

Fig. 3: Microphotograph of a prototype
time-resolving CMOS image sensor.
Fig. 3 shows one of the time-resolving CMOS
image sensors whose feasibility was tested withthe microscope shown in Fig. 2. The lifetimes ofseveral fluorophores such as Coumarine 6 andFlourescein were measured and compared withthe reference values.
(３－２) Ripple effects and further developments
The time-resolving CMOS image sensors are
suitable for the implementation of multiplexed
confocal FLIM, which enhances the throughput ofdrug discovery. This technology will give a great
impact in the field of pharmacy, and happiness ofhuman being.
［４］Achievements (List of Publications)No publication this year

Travelling Report（Mention each travel by CRP budget.）None