Anti-Stokes Fluorescence Spectra of Chloroplasts in Parachlorella kessleri and Maize at Room Temperature as Characterized by Near-Infrared Continuous-Wave Laser Fluorescence Microscopy and Absorption Microscopy

Abstract

Microscopic autofluorescence spectral imaging of chloroplasts in maize mesophyll cells using near-infrared laser excitation has previously shown that a photosystem I spectral component exhibits an intensity similar to that of photosystem II at ~294 K when a continuous-wave laser at 800-820 nm is used. To establish the generality of this phenomenon, chloroplasts in Parachlorella kessleri cells (P. kessleri) were studied. A continuous-wave laser at 785 nm promoted photosystem-I-specific fluorescence in P. kessleri chloroplasts. The difference in chlorophyll fluorescence peak wavelengths between P. kessleri and maize correlated well with those observed at cryogenic temperatures. To further clarify the nature of anti-Stokes fluorescence, we studied chloroplasts in acetone-treated P. kessleri cells (in a medium containing 15% (v/v) acetone) by microscopic fluorescence and absorption spectra on a cell-by-cell basis. A continuous-wave laser at 785 nm led to significant fluorescence from acetone-treated cells, which was attributed to aggregation of chlorophylls. Anti-Stokes fluorescence spectral imaging thus seems to be effective for detection of lowest-energy trap states that are only weakly fluorescent.

Growth‐phase dependent morphological alteration in higher plant thylakoid is accompanied by changes in both photodamage and repair rates

“…Autofluorescence spectra of single chloroplasts were measured by the following two types of near‐infrared excitation lasers. Sub‐picosecond pulses at 808 nm induced fluorescence spectra that predominantly contained PSII and its closely associated light‐harvesting complexes, as shown previously (Hasegawa et al, 2010, 2011). In contrast, CW excitation at 785 nm preferentially excited PSI and its closely associated light‐harvesting complexes, as previously demonstrated in chloroplasts of C 3 and C 4 plants (Hasegawa et al, 2010, 2011; Kim et al, 2015).…”

“…Microscopic fluorescence spectroscopy based on near‐infrared lasers was described previously (Hasegawa et al, 2010, 2011; Kumazaki et al, 2007). A pulsed laser at 808 nm with a pulse duration of 0.2 ps was generated at a repetition frequency of 75.5 MHz from a laser oscillator (Mira, Coherent, Inc.) for two‐photon excitation of photosynthetic pigments.…”

Protein expression guided chemical profiling of living cells by the simultaneous observation of Raman scattering and anti-Stokes fluorescence emission

“…Despite several scattered reports on the anti-Stokes fluorescence emission from chloroplasts25, organic2627 and inorganic dyes28, to the best of our knowledge, the anti-Stokes fluorescence excitation of FPs has never been reported before. To verify whether the anti-Stokes emission from FPs can be detected under Raman measuring conditions, we recorded the emission spectra of various FPs with a shorter emission peak than the continuous-wave (cw) 532 nm laser that we use for our Raman experiments (Fig.…”

Radical Scavenging Activity Assay and Red Fluorescence Microscopy Studies: Antioxidant Properties of Selected Young and Mature Leaves for Application in Pharmaceutical Industry

“…Upon UV light excitation, the various compounds bound in the cells would emit different fluorescence wavelengths. Typically, the blue and green fluorescence have been attributed to ferulic and hydroxycinnamic derivatives, which are bound to the cell wall or mesophyll, whereas red fluorescence has been assigned to be due to the chlorophylls inside the chloroplast [20][21][22][23] . By ImageJ software, the distribution of RGB fluorescence was obtained and the red fluorescence intensity was considered as an indicative of the antioxidant activity.…”