Aleksandr V. Smirnov

Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector

We have constructed a device that maximizes the probability of collecting all of the scattered and ballistic light isotropically generated at the focal spot of multiphoton excited emissions (MPE) to optimize the signal‐to‐noise ratio (SNR) for micro‐imaging. This was accomplished by optically coupling a parabolic reflector (that surrounds the sample and top of the objective) to a pair of collimating lenses (above the sample) that redirects emitted light to a separate detector. These additional optics, combined with the objective, allow the total emission detection (TED) condition to be approached. Numerical simulations suggest an approximately 10‐fold improvement in SNR with TED. Comparisons between the objective detection and TED reveal an enhancement of 8.9 in SNR (77% of predicted) for GFP‐labelled brain slices and similar results for fluorescent beads. This increase in SNR can be used to improve time resolution, reduce laser power requirements/photodynamic damage, and, in certain cases, detection depth, for MPE imaging techniques.

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy.

Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED)

A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction‐limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed [Combs, C.A., et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J. Microsc. 228, 330–337] that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the excitation spot for tissue blocks, leading to ∼8‐fold signal gain. Using a similar approach, we have developed an in vivo total emission detection (epiTED) instrument modified to make noncontact images from outside of living tissue. Simulations suggest that a ∼4‐fold enhancement may be possible (much larger with lower NA objectives than the 0.95 NA used here) with this approach, depending on objective characteristics, imaging depth and the characteristics of the sample being imaged. In our initial prototype, 2‐fold improvements were demonstrated in the mouse brain and skeletal muscle as well as the rat kidney, using a variety of fluorophores and no compromise of spatial resolution. These results show this epiTED prototype effectively doubles emission signal in vivo; thus, it will maintain the image signal‐to‐noise ratio at two times the scan rate or enable full scan rate at approximately 30% reduced laser power (to minimize photo‐damage).

Quantitative detection of the ligand-dependent interaction between the androgen receptor and the co-activator, Tif2, in live cells using two color, two photon fluorescence cross-correlation spectroscopy

Two-photon, two-color fluorescence cross-correlation spectroscopy (TPTCFCCS) was used to directly detect ligand-dependent interaction between an eCFP-fusion of the androgen receptor (eCFP-AR) and an eYFP fusion of the nuclear receptor co-activator, Tif2 (eYFP-Tif2) in live cells. As expected, these two proteins were co-localized in the nucleus in the presence of ligand. Analysis of the cross-correlation amplitude revealed that AR was on average 81% bound to Tif2 in the presence of agonist, whereas the fractional complex formation decreased to 56% in the presence of antagonist. Residual AR–Tif2 interaction in presence of antagonist is likely mediated by its ligand-independent activation function. These studies demonstrate that using TPTCFCCS it is possible to quantify ligand-dependent interaction of nuclear receptors with co-regulator partners in live cells, making possible a vast array of structure-function studies for these important transcriptional regulators.

Compact non-contact total emission detection for in vivo multiphoton excitation microscopy

We describe a compact, non‐contact design for a total emission detection (c‐TED) system for intra‐vital multiphoton imaging. To conform to a standard upright two‐photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non‐contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c‐TED device compared to heavily optimized objective‐based emission collection. The best light collection enhancement was seen from murine fat (5×–2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo‐damage, as well as the applicability of this device to other multiphoton imaging methods is discussed.

A novel global fitting algorithm for decay-associated images from fluorescence lifetime image microscopy data

Fluorescence lifetime imaging microscopy is a technique in which the fluorescence lifetime(s) of a fluorophore is measured at each spatially resolvable element of a microscope image. Imaging of fluorescence lifetimes enables biochemical reactions to be followed at each microscopically resolvable location within the cell. FLIM has thus become very useful for biomedical tissue imaging. Global analysis [1] is a method of recovering fluorescence decay parameters from either time-resolved emission spectra to yield Decay-Associated Spectra [2], or equivalently, from FLIM datasets to yield Decay-Associated Images. Global analysis offers a sensitive and non-invasive probe of metabolic state of intracellular molecules such as NADH. Using prior information, such as the spatial invariance of the lifetime of each fluorescent species in the image, to better refine the relevant parameters, global analysis can recover lifetimes and amplitudes more accurately than traditional pixel-by-pixel analysis. Here, we explain a method to analyze FLIM data so that more accurate lifetimes and DAIs can be computed in a reasonable time. This approach involves coupling an iterative global analysis with linear algebraic operations. It can be successfully applied to image, e.g. metabolic states of live cardiac myocytes, etc.

Fluorescence Studies of Local Environmental Heterogeneity Around Guanine Residues of the Telomeric DNA Quadruplex Conformation

Formation and stabilization of G-quadruplexed DNA formed by the guanine-rich human telomeric sequence d(TTAGGG)4 (HT4), has been shown to inhibit the abnormal activity of telomerase in tumor cells, making stabilization of this DNA secondary structure a target for potential cancer therapeutics. To investigate the effect of local environment on the guanine residues of the G-quadruplex, we previously designed several fluorescence analogs of the HT4 sequence with incorporation of 6-methyl-8-(2-deoxy-D-ribofuranosyl) isoxanthopterin (6MI) at varying guanine-tetrad positions: G1, G4, and G11, which show fluorescence sensitivity to quadruplex folding. In the current studies, we have collected decay-associated spectra (DAS) for the fluorescence lifetime components (τlong, τmedium, and τshort) resolved for each of the mono-labeled sequences. Multi-exponential decay profiles suggest local environmental heterogeneity surrounding the guanine residues in both the folded and unfolded states. Interestingly, the DAS for τlong observed for guanine positions G1 and G11, which are located at the 5’- and 3’-ends, respectively, of the quadruplex-forming sequence, showed significant (10nm) red-wavelength shifts on quadruplex folding. In contrast no DAS shifts are observed for the G4 position, located in the loop region of the folded quadruplex. Thus, in addition to base stacking of the guanine residues with neighboring bases, guanine positions located at G1 and G11, on average, may also be exposed to a more polar environment with quadruplex folding, possibly due to conformational heterogeneity as they are located near the termini of the telomeric sequence. The solvent accessibility of these positions may make them key docking sites for designing ligands that can stabilize the folded conformation of the G-quadruplex.This work was supported by NIH SCORE Grant S06-GM076168-01 (LD).

Probing Variations In The Structural Environment Of A DNA Sequence Using Fluorescence Properties Of The Pteridine Analog Probes, 3MI and 6MI

We explored two different microenvironments in the sequence; 5′-actaGagatccctcagacccttttagtcagtGtgga -3′ in single and duplex form using two similar nucleoside analogs. 3MI and 6MI were each investigated in two different environments, one flanked by thymines (PTRT) and the other, by adenines (PTRA)(shown by Gs noted above). Each site is equidistant from a terminus. The probes differ only by the position of a methyl group in either the 3- (3MI) position or the 6- (6MI) position. Both time-resolved anisotropies and lifetimes of the probes depend upon local electrostatics which are impacted by duplex formation. 3MI shows less response to structural change as compared to 6MI. Integrals of lifetime curves compared with quantum yields of each sample reveal that each displays a “dark” component which we are unable to detect with TCSPC (e.g.,tau<70ps). For 6MI in the A environment this QSSQ “quasi static quenching” eliminates approximately half the molecules, whether in SS or DS form. 6MI in the T environment displays an unexpected increase in the quantum yield upon duplex formation (0.107 to 0.189) apparently the result of escape from QSSQ which simultaneously declines from 66% to 33%. Escape from the dark state is accompanied by doubling of steady state anisotropy of 6MI in PTRT in the duplex. Only 6MI in the T duplex displays a rotational correlation time over 7 ns. The DS A environment fails to constrain local motion and QSSQ remains the same as in SS; in contrast, the flanking T duplex environment restricts local motion and halves the QSSQ.