Ultra-fast frame rate CMOS cameras, combined with a photon counting image intensifier, can be used for microsecond resolution wide-field time-correlated single photon counting (TCSPC) microscopy. A sequence of frames is recorded after an excitation pulse, and the number and location of photons in each frame is determined. This process is repeated until enough photons are recorded for a photon arrival time histogram in the pixels of the image. This approach combines low, nanowatt excitation power with single-photon detection sensitivity and arrival timing in many pixels simultaneously, short acquisition times in the order of seconds and allows lifetime mapping with a time resolution of ~1 microsecond. Moreover, we also show that the phosphor decay can be exploited to time the photon arrival well below the exposure time of the camera. This approach yields better time resolution and larger images than direct imaging of photon events. We show that both methods are ideal for lifetime imaging of transition metal compounds in living cells within a few seconds.
Two-photon fluorescence imaging of proteins labelled with GFP or its analogues provides information on the localization of
the molecules in cells and tissues, and their redistribution on timescales as short as milliseconds. Fluorescence correlation
spectroscopy (FCS) analyzes fluctuations of the fluorescence signal in order to yield information about the motion of the
molecules on timescales considerably shorter than those accessible with imaging, allowing the determination of diffusion
coefficients, estimation of aggregate size, molecular concentrations, etc., i. e., parameters that can be difficult to determine
with imaging alone. Scanning FCS (sFCS) is a modification of FCS that provides information about molecular dynamics
and type of motion, which is too slow for standard FCS, and not resolvable with imaging.
We have applied two-photon imaging, FCS and sFCS to study the localization and redistribution of GFP-labelled proteins
involved in the asymmetric first division of C. elegans embryos. While the distribution of the investigated proteins
in the cytoplasm is homogeneous on the scale limited by the optical resolution and their fast motion can be well characterized
with conventional FCS, the proteins localized in the cortex exhibit patterns evolving on the ms-s temporal scale.
We use sFCS and explore the applicability of spatial correlation analysis (image correlation, STICS) to the qualitative and
quantitative description of the dynamics of the cortex-localized proteins.
Picosecond fluorescence lifetime imaging microscopy (FLIM) provides a most valuable tool to analyze the primary
processes of photosynthesis in individual cells and chloroplasts of living cells. In order to obtain correct lifetimes
of the excited states, the peak intensity of the exciting laser pulses as well as the average intensity has to be
sufficiently low to avoid distortions of the kinetics by processes such as singlet-singlet annihilation, closing of the
reaction centers or photoinhibition. In the present study this requirement is achieved by non-scanning wide-field
FLIM based on time- and space-correlated single-photon counting (TSCSPC) using a novel microchannel plate
photomultiplier with quadrant anode (QA-MCP) that allows parallel acquisition of time-resolved images under
minimally invasive low-excitation conditions. The potential of the wide-field TCSPC method is demonstrated by
presenting results obtained from measurements of the fluorescence dynamics in individual chloroplasts of moss
leaves and living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.
Multi-dimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterised by its time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multi-dimensional TCSPC makes a fluorescence lifetime technique with multi-wavelength capability, near-ideal counting efficiency, and the capability to resolve multi-exponential decay functions. We show that the same technique and the same hardware can be used to for precision fluorescence decay analysis, fluorescence correlation spectroscopy (FCS), and fluorescence intensity distribution analysis (FIDA and FILDA) in selected spots of a sample.
As a precursor to applying fluorescence lifetime imaging (FLIM) to studies of intercellular communication in molecular immunology, we have investigated the fluorescence lifetime of enhanced green fluorescent protein (EGFP) in mixtures of water and glycerol using time-correlated single photon counting (TCSPC). We find that the EGFP lifetime decreases with increasing glycerol content. This is accounted for quantitatively by the refractive index dependence of the fluorescence lifetime as predicted by the Strickler Berg formula which relates the fluorescence lifetime to the absorption spectrum. The solvent viscosity has no influence on the fluorescence lifetime. We also discuss the refractive index dependence of the GFP fluorescence lifetime in more complex systems. The findings are particularly relevant for the interpretation of FLIM of GFP expressed in environments such as bacteria and cells.
Steady state and time resolved confocal fluorescence microscopy, using a point scanning system, is applied to an investigation of the early stages of photo-induced changes in 3T3-L1 murine fibroblasts using di-sulphonated aluminum phthalocyanine (AlPcS2) as a photosensitizer. A comparison is made with data obtained using a line scan system and V79-4 Chinese hamster fibroblasts. The steady state data obtained in this work demonstrate that intracellular AlPcS2 fluorescence intensity increases progressively on photoirradiation. Time-resolved studies indicate that this could result from a progressive decrease in the concentration of the self-quenched membrane-associated form of AlPcS2 following its conversion into the fluorescent monomeric form.
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