UV Microscopy and Spectroscopy

Performing measurements using ultraviolet (UV) light gives researchers access to a range of fluorescent molecules and wide band gap semiconductors. In addition, Raman measurements can benefit from UV excitation through increased signal or resonant enhancement. In some cases, the enhancement can be several orders of magnitude. For these and many other reasons, working with UV is attractive. However, the benefits come with a unique set of challenges. This tutorial describes some of the challenges inherent in UV work and mitigation strategies.

What is UV?

The most basic definition of ultraviolet light is “beyond the violet”. But when it comes to more precise definitions, what exactly qualifies as “ultraviolet” depends on context. Some include 405 nm as UV, while others consider anything shorter than 400 nm as UV. For this article, UV will be considered to be wavelengths 400 nm and shorter. Near UV often refers to UV wavelengths longer than 300 nm, while deep UV refers to wavelengths shorter than 300 nm. Vacuum UV refers to wavelengths shorter than 192 nm. The terms UVA, UVB and UVC (“germicidal UV”) are not commonly used in UV optics or spectroscopy though they often appear in other contexts.

The UV covers a large range of energies. If we consider UV to refer to the range of 400-121 nm, then we have an energy range of 3.10 to 10.25 eV – a span of 7.15 eV. For comparison, the entire visible range only has a span of 1.45 eV. Consequently, the wavelength dependence of nearly all components of an optical system needs to be considered when working in the UV.

Safety

The dangers of UV should not be underestimated. Due to the inability of the human eye to see UV photons, exposure can occur without obvious symptoms. UV photons usually do not penetrate to the retina, but are instead absorbed in the cornea and lens of the eye. Long term exposure can lead to cataracts and other eye problems. Many polycarbonate-based laser eyewear products have a good optical density in the UV and provide excellent protection.

A simple method of finding stray UV beams is to use an item with strong fluorescence, such as some business cards or fluorescent paper, to visualize the beam. It is advisable to trace the beam path from the source all the way through your system. Some laser sources, such as SHG lasers, can have extra emissions that exit at an angle relative to the main beam. These emissions have to be controlled separately.

Black may not be black

A common mistake when working in the UV region is assuming that anything that is black in the visible will also be black in the UV. As convenient as it would be, this assumption is often not true. Since UV photons have higher energies than their visible counterparts, UV photons can excite fluorescence in many materials through either direct excitation or multiphoton absorption.

For example, black anodized aluminum is commonly used in optical systems to help absorb scattered light. However, under UV illumination anodized aluminum has a strong luminescence background that can interfere with measurements. The figure shows spectra and a photograph of black anodized aluminum under UV excitation. It is clear that even this specialized “optically black” coating is not suitable for UV work.

Many paints also contain pigments that become emitters above certain energies. For example, paints often contain ZnO as part of their formula. The band gap of ZnO is 3.4 eV (360 nm), so black paint containing ZnO will become an emitter under common UV excitations such as 355 nm and 325 nm. Other pigments and the binding agents found in paint can luminesce under UV excitation.

In addition, brass and other copper alloys can form a surface coating that will luminesce under UV excitation. Thus, besides their primary properties, the aging of materials also has to be considered. Two materials that can be used are 430 stainless steel and graphite. Both of these typically have low background and are stable over time. Furthermore, UV illumination will often bleach anodized aluminum coatings, reducing their effectiveness as absorbers in the visible range.

Clear may not be clear

Conversely, it is often assumed that things that are clear in the visible – non-absorbing – are also clear in the UV. In reality there are only a few glasses suitable for UV use. Due to the high energies of UV photons, band gap luminescence can be excited along with various defect emissions. A visibly clear piece of material can be an emitter or absorber under UV excitation.

Even in the absence of band edge or defect emission, many materials display larger absorption coefficients in the UV region compared to the visible. Consequently, materials are not as “clear” to the UV as they are to the visible. BK7 and UV grade fused silica are two common glasses that are used in UV applications. For deep and mid UV applications, UV grade fused silica is often the only practical option. Fused silica comes in multiple grades. While most fused silica is sufficiently transparent for visible applications, high quality fused silica – referred to as “UV grade” – is required for UV applications. Other UV-compatible substrates exist, but often suffer from additional difficulties such as strong birefringence or high cost (e.g., fluorite or sapphire based optics) are thus used only in highly specialized applications.

These phenomena can become a particular problem with subsystems such as cameras. For example, a CMOS image sensor will often have a protective piece of glass cemented above the sensor. If this glass is not transparent in the UV, or displays fluorescence, the camera may not be able to see the UV signal or may produce spurious signals. Photoluminescence (PL) and absorption spectroscopies are excellent ways of determining the optical properties of the materials in question. In general, UV systems must be carefully characterized.

Solarization

One of the less-known phenomena associated with UV optics is known as “solarization”. Solarization of glasses occurs when O-H bonds in glasses are broken by UV light, causing a darkening of the glass. This is internal damage to the glass, not merely damage to any coating or surface. Such darkening leads to increased absorption in the glass, therefore reduction of excitation power and reduction in signal. In particular, Ge is sometimes added to glasses to improve their working properties, but Ge doped glasses are much more susceptible to solarization than undoped glasses.

There is no way to entirely eliminate this problem, but proper choice of glasses can reduce the effect. UV grade fused silica has minimal O-H bonds and increased resistance to solarization. Keeping power densities low inside the glass elements assists in delaying solarization. Solarization becomes especially important in fiber optic lines carrying UV photons both due to the high power density and large volume of material through which the photons must pass. Klar uses high quality components to maximize solarization resistance and provides a maintenance plan to keep the instrument at top performance.

Plastics

Nearly all plastics are opaque to UV light or are damaged by UV light. This is because UV photons have sufficient energy to break C-C bonds, thereby damaging and destroying plastics through which they pass. There are a few exceptions—for example, pure FEP (fluorinated ethylene propylene) can be used in some UV applications. As a general rule, however, plastic optical elements should be considered to be opaque to UV light unless specifically designed for UV work.

Plastics appear in two other important contexts: sample mounting and optical cements. In many cases standard mounting methods such as wax, epoxy and various tapes introduce appreciable background signal under UV excitation, potentially leading to spurious signals or overwhelming the desired signal. If at all possible, the ideal is to mount a sample on a metal or other material that does not have an emitting surface oxide, such as certain stainless steels or silicon, or mounted in a way that there is no backing behind the sample at all.

Optical cements are often used in compound lens systems. There are a number of optical adhesives that are reasonably transparent in the near UV, but care must be taken to ensure compatibility.

The strong absorption of plastics can be taken advantage of in the right applications. Using a plastic element can be an excellent means of letting visible light pass but blocking any UV.

Precision of optics

The precision of optics and coatings is often specified in terms of λ/10 – one tenth of a wavelength. Normally the λ refers to a reference wavelength, usually 635 nm or similar, and thus the precision of optics is specified in terms of a comparatively long wavelength. Consider a defect that is λ/10 for 635 nm. This defect is 63.5 nm in size. But for a near-UV wavelength of 317.5 nm, that same defect is now a λ/5 defect, approaching a quarter wavelength of the light. At a deep-UV wavelength of 244 nm, the same defect is λ/3.8. This has important consequences for optical quality and especially for coatings. Since the wavelength of the light passing through a coating is so much shorter than the common reference wavelength, the coating process must be controlled very tightly. Thus, UV optics require extremely high precision. In practice, a precision of λ/3 is common in UV optics where lambda is taken in the UV instead of the 635 nm reference.

Going deep

Since the wavelength dependence of every element of the optical system has to be considered, deep UV applications become increasingly difficult. At wavelengths shorter than 192 nm, even the atmosphere becomes important. This is because oxygen in the air begins to absorb strongly, requiring the optical path to be purged with nitrogen or put under vacuum. For this reason, many commercial UV-Vis absorption instruments have a practical limit of 192 nm, though specialized vacuum UV absorption systems exist.

Objective lenses

Compared to visible applications, there are considerably fewer choices of objective lenses that work well in the UV, and very few have high magnifications. 40× objectives are one of the highest magnifications that are commonly available. High magnification UV objectives exist, but are often extremely expensive. In addition, the exact wavelengths of interest must be specified when shopping for objectives. For the materials reasons stated above, a standard objective listed as transparent at 400 nm may not be transparent at 355 nm, and will probably not be transparent at 325 nm or shorter.

Reflective objectives are often a more budget-friendly choice. Reflective objectives also have certain wavelength preferences, though they are less restrictive than for refractive objectives. Silver is not suitable for some UV work while high-grade aluminum is. Care must be taken, however, as reflective objectives usually have a larger diameter than their refractive counterparts.

Cleaning

When cleaning UV optics, care must be taken to avoid leaving residues or scratches on the surfaces. In general, the techniques are the same as for visible optics, but the quality of the solvents and any air dusters must be taken into account. In general, solvents that have been stored in plastic containers or have been stored where they can absorb contaminants from the atmosphere are unsuitable for cleaning UV optics due to the residues left behind. We recommend use of carefully stored solvents from pre-rinsed containers. Likewise, air dusters can leave difficult to remove residues from the bitterants and oils in the gas stream. Air dusters from office supply stores are not suitable for cleaning of UV optics. Scientific supply companies can provide high purity filtered dusters more suitable for the purpose.

The wiping techniques commonly used for cleaning optics should be used only with extreme care. Because UV wavelengths are much shorter than visible light, even small scratches can be an appreciable fraction of the wavelength of the light. Thus using lens wipes on UV optics should be considered as a last resort and used only after gentle dusting and repeated solvent flooding have failed. Some older systems use uncoated aluminum mirrors. Due to their fragile nature, these optics should never be wiped.

Data density

The efficiency of gratings for spectrometers typically goes down as the impinging wavelengths get shorter. To counter this effect a higher groove density, 2400 lines/mm or greater, is often used in UV spectrometers.

Many modern spectrometers utilize an array detector (e.g., CCD or CMOS) to collect spectral data. Each pixel of the data is a more-or-less constant spatial distance from its neighbors. That is, pixels in the spectra are linear in wavelength, but not linear in energy. But because energy and wavelength are inversely related, the data points spread out on the energy scale as wavelength becomes shorter. This effect is normally unimportant in visible applications but can become significant in the UV region. For example, when performing absorption spectroscopy on a material with a high band gap, the experimenter will often be required to specify a much higher data density than usual to gain enough data points in the main region of interest.

Conclusions

When working with UV light, all elements of the optical system need to be specifically considered and qualified for UV work. This includes the substrates of optical elements, cements, coatings, and sample mounting methods.  Careful attention to materials, performance of individual elements, aging and overall system design can greatly enhance system performance in the UV. Klar has taken care to choose materials and processes that reduce or eliminate sources of background and do not photodegrade to deliver long-term performance. When choosing a Klar instrument you can be confident that every effort has been made to maximize performance in the UV.