Alpha300r confocal raman microscope

Confocal Raman imaging was used for the detection of calcium-phosphate nanoparticle uptake in monocytes. Raman imaging was done with the help of Tom Venus in collaboration with the Institute of Medical Physics and Biophysics, Leipzig University. Monocytes were freshly isolated from human peripheral blood and 1 × 10⁶ monocytes were seeded in Eppendorf cell Imaging dishes (145 µm glass bottom) in RPMI1640 medium to reach ultra-adherence for 60 min (37 °C/5% CO₂). Medium was replaced by 10% FBS containing RPMI1640. Monocytes were imaged before and after adding 2.5 mM [Ca²⁺] with a WiTec alpha300 R+ confocal Raman microscope with an excitation wavelength of 532 nm (34 mW, 50 µm pinhole) and a ×63/1 Zeiss W Plan Apochromat objective under temperature control (38 °C). Raman spectra were collected pixel-wise using a 600 g mM⁻¹ grating, pixel size of 250 µm × 250 µm and an integration time of 70 ms. Images were taken and processed with the WiTec software Control FOUR and Project FOUR PLUS software.

A WITec Alpha300R+ confocal Raman microscope equipped with a Zeiss W Plan-Apochromat VIS-IR 100×/0.9 objective, a 785 nm excitation diode laser (Toptica, Munich, Germany), and an UHTS 300 spectrometer (WiTec, Ulm, Germany) using a −75 °C cooled CCD camera (ANDOR iDus 401, Belfast, UK) was used. A fiber with 100-μm diameter was used as a pinhole.

For Raman spectroscopy measurements,
a WITec alpha300R confocal Raman microscope equipped with a green
laser (532 nm, 20 mW) and a Zeiss Neofluar objective (40× magnification)
were used. The spectra were collected through a 600 g/mm grating with
the spectral center set at 1900 rel. cm^–1 and recorded
with a CCD camera (integration time: 10–20 s). The software
WITec Suite FIVE was employed for cosmic ray removal and background
subtraction.