Global analysis and Decay Associated Images (DAI) derived from Fluorescence Lifetime Imaging Microscopy (FLIM)

Mitchell Harling; Gregory R. Alspaugh; Alessio Andreoni; Aleksander V. Smirnov; Rozhin Penjweini; Michael Murphy; Marie-Paule Strub; Jay R. Knutson

doi:10.1117/12.2514365

22 February 2019 Global analysis and Decay Associated Images (DAI) derived from Fluorescence Lifetime Imaging Microscopy (FLIM)

Mitchell Harling, Gregory R. Alspaugh, Alessio Andreoni, Aleksander V. Smirnov, Rozhin Penjweini, Michael Murphy, Marie-Paule Strub, Jay R. Knutson

Author Affiliations +

Proceedings Volume 10882, Multiphoton Microscopy in the Biomedical Sciences XIX; 108822A (2019) https://doi.org/10.1117/12.2514365
Event: SPIE BiOS, 2019, San Francisco, California, United States

Abstract

The extraction of fluorophore lifetimes in a biological sample provides useful information about the probe environment that is not readily available from fluorescence intensity alone. Cell membrane potential, pH, concentration of oxygen ([O₂]), calcium ([Ca²⁺]), NADH and other ions and metabolites are all regularly measured by lifetime-based techniques. These measurements provide invaluable knowledge about cell homeostasis, metabolism and communication with the cell environment. Fluorescence lifetime imaging microscopy (FLIM) produces spatial maps with time-correlated singlephoton counting (TCSPC) histograms collected and analyzed at each pixel, but traditional TCSPC analysis is often hampered by the low number of photons that can reasonably be collected while maintaining high spatial resolution. More important, traditional analysis fails to employ the spatial linkages within the image. Here, we present a different approach, where we work under the assumption that mixtures of a global set of lifetimes (often only 2 or 3) can describe the entire image. We determine these lifetime components by globally fitting precise decays aggregated over large spatial regions of interest, and then we perform a pixel-by-pixel calculation of decay amplitudes (via simple linear algebra applied to coarser time-windows). This yields accurate amplitude images (Decay Associate Images, DAI) that contain stoichiometric information about the underlying mixtures while retaining single pixel resolution. We collected FLIM data of dye mixtures and bacteria expressing fluorescent proteins with a two-photon microscope system equipped with a commercial single-photon counting card, and we used these data to benchmark the gDAI program.

Citation Download Citation

Mitchell Harling, Gregory R. Alspaugh, Alessio Andreoni, Aleksander V. Smirnov, Rozhin Penjweini, Michael Murphy, Marie-Paule Strub, and Jay R. Knutson "Global analysis and Decay Associated Images (DAI) derived from Fluorescence Lifetime Imaging Microscopy (FLIM)", Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108822A (22 February 2019); https://doi.org/10.1117/12.2514365

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available

Members: $17.00

Non-members: $21.00 ADD TO CART

PROCEEDINGS
16 PAGES

DOWNLOAD PAPER SAVE TO MY LIBRARY

GET CITATION

RIGHTS & PERMISSIONS

Get copyright permission Get copyright permission on Copyright Marketplace

KEYWORDS

Fluorescence lifetime imaging

Photons

Bacteria

Luminescence

Microscopy

Image analysis

Fluorescent proteins

Show All Keywords

Keywords/Phrases

Search In:

Publication Years