Photodynamic therapy (PDT) of cancer is dependent on three primary components: photosensitizer (PS), light, and oxygen. Because these components are interdependent and vary during the dynamic process of PDT, assessing PDT efficacy may not be trivial. Therefore, it has become necessary to develop pre-treatment planning, on-line monitoring and dosimetry strategies during PDT, which become more critical for two or more chromophore systems, e.g. PS-CD conjugates developed in our laboratory for fluorescence-imaging and PDT of cancer. In this study, we observed a significant impact of variable light dosimetry; (i) high light fluence and fluence rate (light dose: 135 J/cm², fluence rate: 75 mW/cm²) and (ii) low light fluence and fluence rate (128 J/cm² and 14 mW/cm² and 128 J/cm² and 7 mW/cm²) in photobleaching of the individual chromophores and their long-term tumor response. The fluorescence at the near-infrared (NIR) region of the PS-NIR fluorophore conjugate was assessed intermittently via fluorescence imaging. The loss of fluorescence, photobleaching, caused by singlet oxygen from the PS was mapped continuously during PDT. The tumor responses (BALB/c mice bearing Colon26 tumors) were assessed after PDT by measuring tumor sizes daily. Our results showed distinctive photobleaching kinetics rates between the PS and CD. Interestingly, compared to higher light fluence, the tumors exposed at low light fluence showed reduced photobleaching and enhanced long-term PDT efficacy. The presence of NIR fluorophore in PS-CD conjugates provides an opportunity of fluorescence imaging and monitoring the photobleaching rate of the CD moiety for large and deeply seated tumors and assessing PDT tumor response in real-time.

Trace metals are essential elements that play key roles in a number of biochemical processes governing human visual physiology in health and disease. Several trace metals, such as zinc, have been shown to play important roles in the visual phototransduction process. In spite of this, there has been little research conducted on the direct effect of trace metal elements on the visual pho-toreceptor rhodopsin. In the current study, we have determined the effect of several metal ions, such as iron, copper, chromium, manganese, and nickel on the conformational stability of rho-dopsin. To this aim, we analyzed, by means of UV-visible and fluorescence spectroscopic methods, the effects of these trace elements on the thermal stability of dark rhodopsin, the stability of its active Metarhodopsin II conformation, and its chromophore regeneration. Our results show that copper prevented rhodopsin regeneration and slowed down the retinal release process after il-lumination. In turn, Fe3+, but not Fe2+, increased the thermal stability of the dark inactive con-formation of rhodopsin, whereas copper ions markedly decreased it. These findings stress the important role of trace metals in retinal physiology at the photoreceptor level, and may be useful for the development of novel therapeutic strategies to treat retinal disease.

Optical tissue imaging has several advantages over the routine clinical imaging methods, including non-invasiveness (does not change the structure of tissues), remote operation (avoids infection) and ability to quantify the tissue condition by means of specific image parameters. Dermatologists and other skin experts need compact (preferably pocket-size), self-sustained and easy-to-use imaging devices. The operational principles and designs of ten portable in-vivo skin imaging prototypes developed at the Biophotonics Laboratory of Institute of Atomic Physics and Spectroscopy, University of Latvia during the recent five years are presented in this paper. Four groups of imaging devices are considered. Multi-spectral imagers offer possibilities for distant mapping of specific skin parameters, so facilitating better diagnostics of skin malformations. Autofluorescence intensity and photobleaching rate imagers show a promising potential for skin tumor identification and margin delineation. Photoplethysmography video-imagers ensure remote detection of cutaneous blood pulsations and can provide real-time information on cardiovascular parameters and anesthesia efficiency. Multimodal skin imagers perform several of the above-mentioned functions by taking a number of spectral and video images with the same image sensor. Design details of the developed prototypes and results of clinical tests illustrating their functionality are presented and discussed.

Amorphous chalcogenide (ChGs) glasses are intrinsically metastable, highly photosensitive, and therefore exhibit numerous lightinduced effects upon bandgap and sub-bandgap illumination. Depending on the pulse duration of the excitation laser, ChGs exhibit a series of lightinduced effects spanning over femtosecond to seconds time domain. For continuous wave illumination, the effects are dominantly metastable in terms of photodarkening (PD) and photobleaching (PB) that takes place via homopolar to heteropolar bond conversion. On the other hand, under nanosecond and ultrafast pulsed illumination, ChGs exhibit transient absorption (TA) that is instigated from the transient bonding rearrangements through self-trapped exciton recombination. In the first part of the review, we pay special attention to continuous wave lightinduced PD and PB, while in the second part we will focus on the TA and controlling such effects via internal and external parameters e.g., chemical composition, temperature, sample history etc.

We have investigated the canine of olive flounder in terms of the nonlinear optical signals of two-photon excitation autofluorescence (2PEF) and second harmonic generation (SHG), whereby two kinds of nonlinear microscope images of the canine were obtained using ~140 femtosecond pulses. For increasing excitation power of laser pulses, a photobleaching effect was found to be significant in the red and green ranges of autofluorescence, where a double-exponential decay of the intensity occurs within hundreds of second due to collagen fluorophores in the canine. In the blue range of autofluorescence, the photobleaching is suppressed due to an energy conversion of the incident laser pulse to SHG signal. As a result, the signal in blue range remains constant for long term exposure to laser. We have also found that the SHG intensity depends on the orienta-tion of linearly polarized excitation, and the optical anisotropy of SHG can be attributed to the collagen alignment of canines.

Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE) where its fluorescence properties are used to assess the retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE, and lipofuscin-laden-cells to visible light, and determine whether an abundant component of lipofuscin, docosahexaenoate (DHA) can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible leads to a decrease of its long-wavelength fluorescence at about 610 nm with concomitant growth of the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure to light of lipofuscin-laden cells leads to loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes of fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra together with quantitation of intensity of long-wavelength fluorescence can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, thereby useful for screening the retina for increased oxidative damage and early AMD-related changes.

The biophysical consequences of nanoscale curvature have been challenging to resolve due to size-dependent membrane behavior and the experimental resolution limits imposed by optical diffraction. Recent advances in nanoengineering and super-resolution techniques have enabled new capabilities for creating and observing curvature. In particular, draping supported lipid bilayers over lithographically patterned substrates provides a model system for endocytic pits. The experiments and simulations presented below describe the possible detection of membrane curvature through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), single particle tracking (SPT), and polarized localization microscopy (PLM). FRAP and FCS depend on diffraction-limited illumination and detection. In particular, a simulation of FRAP shows no effects on lipids diffusion due to a 50 nm diameter membrane bud at any stage in the budding process. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was amplified with curvature-dependent lipid mobility changes. However, PLM and SPT achieve sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of curvature on lipid behavior have been resolved.