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單分子檢測(cè)水平的定量細(xì)胞生物學(xué)檢測(cè)儀器。

受激發(fā)射損耗（STED）是一種強(qiáng)大的顯微技術(shù)，可以觀察到空間分辨率低于衍射極限的熒光結(jié)構(gòu)。Alba-STED使用脈沖激發(fā)和脈沖耗盡方法（pSTED）結(jié)合數(shù)字頻域熒光壽命成像（FastFLIM）來(lái)記錄時(shí)間分辨光子，從而提高圖像分辨率并分離具有相同激發(fā)波長(zhǎng)的兩個(gè)熒光標(biāo)記物。

關(guān)鍵特色 ： Alba-STED for FLIM/FFS:

• pSTED (Pulsed excitation and pulsed STED)

• FastFLIM for time-resolved pSTED acquisition

• Improved image resolution using the phasor plot

• Dual-label excitation

• Fast image acquisition (dwell time: 0.2 μs)

• High dynamic range (signal up to 60 million counts/s)

測(cè)試實(shí)例展示 Alba-STED for FLIM/FFS

Confocal (left) vs. pSTED (right) images of the actin labeled with the SiR dye in fixed glia cells， acquired by FastFLIM.

Confocal (left) vs. pSTED (right) images of the actin labeled with the SiR dye in fixed glia cells， acquired by FastFLIM.

Confocal images of 60nm fluorescence beads (left); pSTED images (middle); sharpening the pSTED image using a binary filter based on the phasor plots (right).

Dual labels can be separated using pSTED and FastFLIM. Atto 647N and Atto 655 were used as labels; they both are excited by the 640 nm laser. The two dyes are first separated using the phasor plots， and then assigned with two different false colors (Atto 647N - yellow， Atto 655 - purple) to produce the processed and merged pSTED image of the two labels.

參考論文：

Monomeric cohesin state revealed by live-cell single-molecule spectroscopy.

Liu， W.， Biton， E.， Pathania， A.， Matityahu， A.， Irudayaraj， J.， Onn， I.

EMBO Rep. 2019 Dec 29:e48211. doi: 10.15252/embr.201948211. [Epub ahead of print]

Photon-separation to enhance the spatial resolution of pulsed STED microscopy.

Tortarolo， G.， Sun， Y.， Teng， K.W.， Ishitsuka， Y.， Lanzanó， L.， Selvin， P.R.， Barbieri， B.， Diaspro， A.， Vicidomini， G.

Nanoscale. 2019 Jan 9. doi: 10.1039/c8nr07485b. [Epub ahead of print]

A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses.

Wang， R.， Brustlein， S.， Mailfert， S.， Fabre， R.， Fallet， M.， Sivankutty， S.， Rigneault， H.， Marguet， D.

Methods. 2018 May 1;140-141:212-222. doi: 10.1016/j.ymeth.2018.02.010. Epub 2018 Feb 14.

A Novel Pulsed STED Microscopy Method Using FastFLIM and the Phasor Plots

Sun， Y.，Tortarolo G.， Teng， K.-W.， Ishitsuka， Y.， Coskun， U.C.， Liao， S.-C.， Diaspro， A.， Vicidomini， G.， Selvin， P.R.， Barbieri， B.

Proc. SPIE 10069， Multiphoton Microscopy in the Biomedical Sciences XVII， 100691C (February 21， 2017)