Absorption lines occur when an atom, element or molecule absorbs a photon with an energy equal to the difference between two energy levels. This causes an electron to be promoted into a higher energy level, and the atom, element or molecule is said to be in an excited state.

When its electron jumps from higher energy level to a lower one, it releases a photon. Those photons cause different colours of light of different wavelengths due to the different levels. Those photons appear as lines.

The main difference between emission and absorption spectra is that an emission spectrum has different coloured lines in the spectrum, whereas an absorption spectrum has dark-coloured lines in the spectrum.

key concepts and summary When electrons move from a higher energy level to a lower one, photons are emitted, and an emission line can be seen in the spectrum. Absorption lines are seen when electrons absorb photons and move to higher energy levels.

Those same wavelengths appear in emission when the gas is observed at an angle with respect to the radiation source. Why do atoms absorb only electromagnetic energy of a particular wavelength? Thus, each spectral line corresponds to one particular transition between energy states of the atoms of a particular element.

Emission is the process of elements releasing different photons of color as their atoms return to their lower energy levels. Atoms emit light when they are heated or excited at high energy levels. Absorption occurs when electrons absorb photons which causes them to gain energy and jump to higher energy levels.

An atomic emission spectrum is the pattern of lines formed when light passes through a prism to separate it into the different frequencies of light it contains. Each of these spectral lines corresponds to a different electron transition from a higher energy state to a lower energy state.

The emission maximum is chosen and only emission light at that wavelength is allowed to pass to the detector. Excitation is induced (usually by means of a monochromator) at various excitation wavelengths and the intensity of the emitted fluorescence is measured as a function of wavelength.

In an excitation spectrum, the emission monochromator is set to some wavelength where the sample is known to emit radiation and the excitation monochromator is scanned through the different wavelengths.

A fluorophore is excited most efficiently by light of a particular wavelength. This wavelength is the excitation maximum for the fluorophore. This wavelength is the emission maximum for that fluorophore. The excited fluorophore can also emit light at wavelengths near the emission maximum, as shown.

That is the reason why the excitation light normally has a shorter wavelength (i.e., higher energy) than the emission light (i.e., lower energy). In many fluorochromes, the same electronic transitions are involved in both excitation and emission, which leads to near-mirror image spectra.

Mirror image rule: emission spectra are mirror images of the. lowest energy absorption band. A – photon absorption.

The transitional energy that is either absorbed or emitted is in the form of light and so an ICP Spectrometer measures the wavelength of light emitted by an atom and an Atomic Absorption Spectrometer measures the wavelength of light absorbed by an atom, during transitional energy changes. …

There are many different approaches for measuring absorption spectra. The most common one is to point a generated beam of light at a sample and detect the intensity of the radiation that goes through it. The energy that is then transmitted is used to calculate the absorption.

Fluorophores absorb a range of wavelengths of light energy, and also emit a range of wavelengths. Because the excitation and emission wavelengths are different, the absorbed and emitted light are detectable as different colors or areas on the visible spectrum.

Typical fluorescent minerals include: aragonite, apatite, calcite, fluorite, powellite, scheelite, sodalite, willemite, and zircon. But almost any mineral can “glow” under UV light with the right conditions. Most pure minerals do not fluoresce (certain minerals such as scheelite are exceptions).

DAPI (4′,6-diamidino-2-phenylindole) is a blue-fluorescent DNA stain that exhibits ~20-fold enhancement of fluorescence upon binding to AT regions of dsDNA. It is excited by the violet (405 nm) laser line and is commonly used as a nuclear counterstain in fluorescence microscopy, flow cytometry, and chromosome staining.

Hoechst and DAPI are popular blue fluorescent, nuclear-specific dyes that can be used to stain live or fixed cells. The dyes have minimal fluorescence in solution, but become brightly fluorescent upon binding to DNA. The staining is very stable and non-toxic to live cells for several days or longer.

DAPI (4′,6-diamino-2-phenylindole, dihydrochloride) is a fluorescent nucleic acid stain that binds to minor grove A-T rich regions of double-stranded DNA. It is essentially excluded from viable cells, but can penetrate cell membranes of dead or dying cells.

DAPI is a known mutagen and should be handled with care.

In experiments, DAPI has high cytotoxicity, which reinforces the reason to avoid using DAPI for live cell staining. DAPI is fairly non-toxic to humans if exposure occurs.

DAPI staining of nuclei also allows one to identify the nucleolus, which appears as a black cavity in the nucleus due to a threefold lower concentration of DNA in the nucleolus compared to the surrounding nucleoplasm (excluding centromeres) (Figure 1A; see fluorescence intensity plot).

DAPI is a dye that can be used as a tool to visualize nuclear changes and assess apoptosis. Additionally, apoptotic cells stained with DAPI may have observable nuclear blebbing which may help in differentiating necrotic cells which do not blebb.

TUNEL assays involve the application of a nuclear stain to identify cells, followed by a TUNEL label which detects DNA double-strand breaks. The method works thanks to the ability of the TdT enzyme to label blunt ends of double-stranded DNA, allowing the unique nuclear events of apoptosis to be directly visualized.

Apoptosis is detected by measuring the externalization of phosphatidylserine on the plasma membrane using fluorescent-tagged annexin V. Additionally, flow cytometry can be employed to determine alterations in cell size (Bortner and Cidlowski, 2001; Warnes et al. 2011).

An EM finding typical of cells of malignant lymphomas, which consists of the loss of coherence of the nuclear membrane with the nucleoplasm.